Tumor-specific T cell tolerance represents one of the major challenges in cancer immunotherapy. To elicit effective anti-tumor immunity, it is necessary to develop immunotherapeutic strategies capable of overcoming T cell tolerance. In a murine model of A20 lymphoma, we have demonstrated that tumor-specific CDS tolerance is mediated by CD4+CD25+ regulatory T (TReg) cells and that reversal of established TReg cell mediated tumor-specific CDS tolerance by tumor vaccines depends on sustained Toll-like receptor (TLR) signals of innate immunity in vivo. This can be achieved by virus-based vaccines that can provide TLR signals intrinsically or mature DC vaccines with co-administration of a TLR ligand. More importantly, provision of TLR signals significantly enhances the efficacy of tumor vaccines in treating pre-established A20 lymphoma, suggesting an essential role of TLR signals in cancer immunotherapy. In this application, we will investigate the mechanisms underlying TLR-dependent reversal of TReg cell-mediated tumor-specific CDS tolerance in vivo. Specifically, we will pursue the following five aims: 1) To determine if reversal of TReg cell-mediated tumor-specific CDS tolerance in vivo by vaccinia virus vs. adenovirus vaccines is mediated through distinct TLR pathways; 2) To delineate the role of pro-inflammatory cytokines IL-6, IL-12 and/or IL-1 in reversal of TReg cell-mediated tumor-specific CDS tolerance in vivo; 3)To assess the role of Type I interferons (IFNs) in overcoming TReg cell-mediated tumor-specific CD8 tolerance in vivo; 4) To investigate the role of glucocorticoid-induced TNF receptor family-related protein (GITR) and GITR ligand interaction in breaking TReg cell-mediated tumor-specific CDS tolerance in vivo; 5) To evaluate the efficacy of tumor vaccines with TLR signals in treating pre-established A20 lymphoma. The outcome of these studies should have significant impact on the design of effective immunotherapeutic strategies for treating cancer.